This application is a 371 of PCT/EP99/05063 filed Jul. 15, 1999.
The invention relates to a process for producing capsules with a polyelectrolyte shell, and to the capsules obtainable by the process.
Microcapsules are known in various embodiments and are used in particular for controlled release and targeted transport of active pharmaceutical ingredients, and for protecting sensitive active ingredients such as, for example, enzymes and proteins.
Microcapsules can be produced by mechanical/physical processes, or spraying and subsequent coating, chemical processes such as, for example, interfacial polymerization or condensation or polymer phase separation or by encapsulating active ingredients in liposomes. However, processes disclosed to date have a number of disadvantages.
German patent application 198 12 083.4 describes a process for producing microcapsules with a diameter of  less than 10 xcexcm, where several consecutive layers of oppositely charged polyelectrolyte molecules are applied to an aqueous dispersion of template particles. The template particles described in this connection are, in particular, partially crosslinked melamine-formaldehyde particles. After formation of the polyelectrolyte shell it is possible to disintegrate the melamine-formaldehyde particles by adjusting an acidic pH or by sulfonation.
It has been found, surprisingly, that polyelectrolyte capsules can also be formed with use of templates selected from biological cells, aggregates of biological or/and amphiphilic materials such as, for example, erythrocytes, bacterial cells or lipid vesicles. The encapsulated template particles can be removed by subsequent solubilization or disintegration.
The invention thus relates to a process for producing capsules with a polyelectrolyte shell, where several consecutive layers of oppositely charged polyelectrolyte molecules are applied to a template selected from aggregates of biological or/and amphophilic materials, and the template is subsequently disintegrated where appropriate.
Examples of template materials which can be used are cells, for example eukaryotic cells, such as, for example, mammalian erythrocytes or plant cells, single-cell organisms such as, for example, yeasts, bacterial cells such as, for example, E.coli cells, cell aggregates, subcellular particles such as, for example, cell organelles, pollen, membrane preparations or cell nuclei, virus particles and aggregates of biomolecules, for example protein aggregates such as, for example, immune complexes, condensed nucleic acids, ligand-receptor complexes etc. The process of the invention is also suitable for encapsulating living biological cells and organisms. Likewise suitable as templates are aggregates of amphophilic materials, in particular membrane structures such as, for example, vesicles, for example liposomes or micelles, and other lipid aggregates.
Several oppositely charged polyelectrolyte layers are deposited on these templates. This is done by firstly dispersing the template particles preferably in a suitable solvent, for example an aqueous medium. It is then possiblexe2x80x94especially when the template particles are cells or other biological aggregatesxe2x80x94to add a fixing reagent in sufficient concentration to bring about at least partial fixation of the template particles. Examples of fixing reagents are aldehydes such as formaldehyde or glutaraldehyde, which are preferably added to the medium to a final concentration between 0.1-5% (w/w).
Polyelectrolytes mean in general polymers with groups which are capable of ionic dissociation and may be a constituent or substituent of the polymer chain. The number of these groups capable of ionic dissociation in polyelectrolytes is normally so large that the polymers are water-soluble in the dissociated form (also called polyions). In this connection, the term poly-electrolytes also means ionomers in which the concentration of ionic groups is insufficient for water solubility but which have sufficient charges to enter into self-assembly. The shell preferably comprises xe2x80x9ctruexe2x80x9d polyelectrolytes. Depending on the nature of the groups capable of dissociation, polyelectrolytes are divided into polyacids and polybases.
On dissociation of polyacids there is formation of polyanions, with elimination of protons, which can be both inorganic and organic polymers. Examples of polyacids are polyphosphoric acid, polyvinylsulfuric acid, polyvinylsulfonic acid, polyvinylphosphonic acid and polyacrylic acid. Examples of the corresponding salts, which are also referred to as polysalts, are polyphosphate, polysulfate, polysulfonate, polyphosphonate and polyacrylate.
Polybases contain groups able to take up protons, for example by reaction with acids to form salts. Examples of polybases with groups capable of dissociation located on the chains or laterally are polyallylamine, polyethyleneimine, polyvinylamine and polyvinylpyridine. Polybases form polycations by taking up protons.
Polyelectrolytes suitable according to the invention are both biopolymers such as, for example, alginic acid, gum arabic, nucleic acids, pectins, proteins and others, and chemically modified biopolymers such as, for example carboxymethylcellulose and ligninsulfonates, and synthetic polymers such as, for example, polymethacrylic acid, polyvinylsulfonic acid, polyvinyiphosphonic acid and polyethyleneimine.
It is possible to employ linear or branched polyelectrolytes. The use of branched polyelectrolytes leads to less compact polyelectrolyte multifilms with a higher degree of porosity of the walls. To increase the capsule stability it is possible to crosslink polyelectrolyte molecules within and/or between the individual layers, for example by crosslinking amino groups with aldehydes. A further possibility is to employ amphiphilic polyelectrolytes, for example amphiphilic block or random copolymers with partial polyelectrolyte characteristics to reduce the permeability to small polar molecules. Such amphiphilic copolymers consist of units differing in functionality, for example acidic or basic units on the one hand, and hydrophobic units on the other hand, such as styrenes, dienes or siloxanes etc., which can be arranged as blocks or randomly distributed over the polymer. It is possible by using copolymers which change their structure as a function of the external conditions to control the permeability or other properties of the capsule walls in a defined manner. Suitable examples thereof are copolymers with a poly-(N-isopropylacrylamide) content, for example poly-(N-isopropylacrylamide-acrylic acid), which change their water solubility as a function of the temperature, via the hydrogen bonding equilibrium, which is associated with swelling.
The release of entrapped active ingredients can be controlled via the dissolution of the capsule walls by using polyelectrolytes which are degradable under particular conditions, for example photo-, acid- or baselabile polyelectrolytes. A further possibility for particular possible applications is to use conducting polyelectrolytes or polyelectrolytes with optically active groups as capsule components.
There are in principle no restrictions on the polyelectrolytes or ionomers to be used as long as the molecules used have a sufficiently high charge or/and have the ability to enter into a linkage with the underlying layer via other interactions such as, for example, hydrogen bonding and/or hydrophobic interactions.
Suitable polyelectrolytes are thus both low molecular weight polyelectrolytes or polyions and macromolecular polyelectrolytes, for example also polyelectrolytes of biological origin.
For the application of the polyelectrolyte layers to the template there is preferably firstly production of a dispersion of the template particles in an aqueous solution. A polyelectrolyte species with the same or the opposite charge as the surface of the template particle is then added to this dispersion. After removal of any excess polyelectrolyte molecules present, the oppositely charged polyelectrolyte species used to build up the second layer is added. Subsequently there are further alternate applications of oppositely charged layers of polyelectrolyte molecules, it being possible to choose for each layer with the same charge identical or different polyelectrolyte species or mixtures of polyelectrolyte species. The number of layers can in principle be chosen as desired and is, for example, 2 to 40, in particular 4 to 20, polyelectrolyte layers.
After application of the required number of layers, the enveloped template particles canxe2x80x94if desiredxe2x80x94be disintegrated. The disintegration can take place by adding lytic reagents. Lytic reagents suitable in this case are those able to disintegrate biological materials such as proteins or/and lipids. The lytic reagents preferably contain a deproteinizing agent, for example peroxo compounds such as, for example, H2O2 or/and hypochlorite compounds such as, for example, sodium or potassium hypochlorite. The disintegration of the template particles surprisingly takes place within a short incubation time, for example 1 min to 1 h at room temperature. The disintegration of the template particles is substantially complete because even on examination of the remaining shells under the electron microscope there are no longer any residues of the particles detectable. On incorporation of biological polyelectrolytes into the shell it is also possible to produce empty layers within the polyelectrolyte shell.
Capsules obtainable by the process of the invention can be produced with diameters in the range from 10 nm to 50 xcexcm, preferably 50 nm to 10 xcexcm, also in shapes differing from spherical, that is to say anisotropic. The wall thickness is determined by the number of polyelectrolyte layers and can be, for example, in the range from 2 to 100 nm, in particular in the range from 5 to 80 nm. The capsules are also distinguished by their monodispersity, that is to say on selection of suitable templates it is possible to obtain capsule compositions with a proportion of less than 10%, and particularly preferably less than 1%, of capsules whose difference from the average diameter is  greater than 50%.
The capsules are very stable toward chemical, biological, organic and thermal stresses. They can be frozen or freeze-dried and then taken up again in suitable solvents.
Since the capsules represent microimpressions of the templates contained in them, and retain their shape even after removal of the templates, it is possible to produce anisotropic particles which comprise microimpressions of biological structures such as cells, virus particles or biomolecule aggregates.
The permeability properties in the shell can be modified by forming or altering pores in at least one of the polyelectrolyte layers. Such pores may be formed automatically on use of appropriate polyelectrolytes. It is additionally possible to employ nanoparticles with anionic or/and cationic groups or/and surface-active substances such as, for example, surfactants or/and lipids for modifying the permeability and other properties. The permeability can additionally be modified by varying the conditions prevailing on deposition of the polyelectrolytes. Thus, for example, a high salt concentration in the surrounding medium results in a high permeability of the polyelectrolyte shell.
A particularly preferred modification of the permeability of polyelectrolyte shells can be achieved be depositing lipid layers or/and amphiphilic polyelectrolytes on the polyelectrolyte shell after disintegration of the template particles. It is possible in this way to reduce very greatly the permeability of the polyelectrolyte shells for small and polar molecules. Examples of lipids which can be deposited on the polyelectrolyte shells are lipids which have at least one ionic or ionizable group, for example phospholipids such as, for example, dipalmitoylphosphatidic acid or zwitterionic phospholipids such as, for example, dipalmitoylphosphatidylcholine or else fatty acids or corresponding long-chain alkylsulfonic acids. On use of zwitterionic lipids it is possible to deposit lipid multilayers on the polyelectrolyte shell. Further polyelectrolyte layers can subsequently be deposited on the lipid layers.
The capsules produced by the process can be used for entrapping active ingredients. These active ingredients may be both inorganic and organic substances. Examples of such active ingredients are catalysts, in particular enzymes, active pharmaceutical ingredients, polymers, dyes such as, for example, fluorescent compounds, sensor molecules, that is to say molecules which react detectably to a change in surrounding conditions (temperature, pH), crop protection agents and aroma substances.
The capsules can also be used as microreaction chambers for chemical reactions or as precipitation or crystallization templates. Because of the fact that the permeability of the capsule walls can be controlled so that, for example, they allow low molecular weight substances to pass through but substantially retain macromolecules, the high molecular weight products resulting from a chemical reaction, for example polymers resulting from a polymerization, can be retained in the interior in a simple way during the synthesis. The reaction product synthesized at the same time in the external medium can be removed subsequently or even during the reaction, for example by centrifugation or/and filtration.
The supply of the reaction substrate can be controlled during the reaction by the diffusion through the capsule walls. New ways of intervening in the progress of reactions emerge from this. Since the external medium can be replaced continuously for example by filtration or else suddenly for example by centrifugation, the polymerization reaction can be stopped as desired by removing the substrate, or the monomer can be replaced. It is thus possible to produce defined copolymers or multipolymers in a novel way. Since the progress of the reaction can be controlled by the monomer supply through the permeation, it is possible to produce in the capsules products with novel and different molecular weight distributions, for example highly monodisperse products. Polymers synthesized inside capsules can be detected, for example, spectroscopically by titration with fluorescent dyes and by confocal microscopy. The gain in mass, and thus the reaction kinetics, can be followed by single-particle light scattering.
On use of anisotropic capsules for packing active ingredients or as reaction chambers, for example for syntheses or precipitation processes, and, where appropriate, subsequent disintegration of the template shells, it is possible to produce particle compositions as dispersions with predetermined shapes and forms. The invention thus also relates to anisotropic particle compositions which are obtainable by encapsulating active ingredients in a polyelectrolyte shell, for example by synthesis or precipitation and subsequent removal of the template, for example by thermal or chemical treatment. These anisotropic particles preferably have the shape of the biostructures used as template.
A further possibility is to use the capsules for introducing organic liquids such as, for example, alcohols or hydrocarbons, for example hexanol, octanol, octane or decane, or for encapsulating gases. Such capsules filled with an organic, water-immiscible liquid can also be employed for chemical reactions, for example polymerization reactions. The monomer can thus be specifically concentrated in the interior of the capsules through its distribution equilibrium. It is possible where appropriate for the monomer solution to be encapsulated in the interior even before the start of the synthesis.
However, it is also possible to encapsulate active ingredients which are unable, because of their size, to penetrate the polyelectrolyte shell. For this purpose, the active ingredient to be entrapped is immobilized on the template particle or is encapsulated by the template particle, for example by phagocytosis or endocytosis in the case of living cells. After disintegration of the template particles, the active ingredient is released inside the polyelectrolyte shell. It is expedient to choose the conditions for disintegration of the template particle in this case so that no unwanted decomposition of the active ingredient takes place.
The capsules can be employed in numerous areas of application, for example sensor systems, surface analysis, as emulsion carriers, microreaction chambers such as, for example, for catalytic processes, polymerization, precipitation or crystallization processes, in pharmacy and medicine, for example for targeting of active ingredients or as ultrasonic contrast agents, in food technology, cosmetics, biotechnology, information technology and the printing industry (encapsulation of dyes). The capsules can further be employed for building up microcomposites or nanocomposites, that is to say materials consisting of at least two different materials and having a microscopic or nanoscopic arrangement.
Yet another aspect of the invention comprises partial disintegration of the template particles preferably in fixed form before the polyelectrolyte coating by treatment with a lytic reagent. Stopping the lytic process at the appropriate time results in partially disintegrated structures, for example toroidal structures with a hole in the middle, which can subsequently be coated. Subsequent complete degradation of the template particles results in annular capsules. This is an entirely novel type of topology with interesting possible applications, for example in optics (micro whispering gallery effect).